WO1993015607A1 - Paires d'ions de composes d'hypericine ayant une activite antivirale - Google Patents

Paires d'ions de composes d'hypericine ayant une activite antivirale Download PDF

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WO1993015607A1
WO1993015607A1 PCT/US1993/001393 US9301393W WO9315607A1 WO 1993015607 A1 WO1993015607 A1 WO 1993015607A1 US 9301393 W US9301393 W US 9301393W WO 9315607 A1 WO9315607 A1 WO 9315607A1
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hypericin
ion pair
accordance
ion
alkyl
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PCT/US1993/001393
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English (en)
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Yehuda Mazur
Daniel Meruelo
Gad Lavie
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Yeda Research And Development Co. Ltd.
New York University
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Priority to JP5514335A priority Critical patent/JPH07504170A/ja
Priority to AU37205/93A priority patent/AU679676B2/en
Priority to EP93906002A priority patent/EP0644869A4/en
Publication of WO1993015607A1 publication Critical patent/WO1993015607A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C50/00—Quinones
    • C07C50/26—Quinones containing groups having oxygen atoms singly bound to carbon atoms
    • C07C50/36—Quinones containing groups having oxygen atoms singly bound to carbon atoms the quinoid structure being part of a condensed ring system having four or more rings
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
    • A01N35/06—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing keto or thioketo groups as part of a ring, e.g. cyclohexanone, quinone; Derivatives thereof, e.g. ketals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
    • C07C211/01—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms
    • C07C211/02—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C215/00—Compounds containing amino and hydroxy groups bound to the same carbon skeleton
    • C07C215/02—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C215/04—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated
    • C07C215/06—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic
    • C07C215/08—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic with only one hydroxy group and one amino group bound to the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/26—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having more than one amino group bound to the carbon skeleton, e.g. lysine
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B3/00—Dyes with an anthracene nucleus condensed with one or more carbocyclic rings
    • C09B3/78—Other dyes in which the anthracene nucleus is condensed with one or more carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B69/00—Dyes not provided for by a single group of this subclass
    • C09B69/02—Dyestuff salts, e.g. salts of acid dyes with basic dyes
    • C09B69/04—Dyestuff salts, e.g. salts of acid dyes with basic dyes of anionic dyes with nitrogen containing compounds

Definitions

  • the present invention relates to ion pairs of hypericin and their use to inhibit the growth of viruses.
  • Hypericin a constituent of plants of the genus
  • Hypericum has been obtained in pure form from plants (Brockman, et al., Ann. 553:1 (1942)), and has also been totally synthesized, (Brockman, et al, Chem. Ber. 90:2302-2310 (1957) and Brockman, et al, Chem. Ber. 90:2480-2491 (1957)).
  • Hypericin both of plant origin and synthetic, has been found to be a potent inhibitor of a wide spectrum of DNA and RNA containing viruses, and particularly of retroviruses, such as Human Immunodeficiency Virus (HIV), the presumed causative agent of AIDS and other conditions.
  • HIV Human Immunodeficiency Virus
  • U.S. Patent No. 4,898,891 issued February 6, 1990, discloses antiviral pharmaceutical compositions containing hypericin, pseudohypericin or pharmaceutically acceptable salts thereof and methods for using these compositions to treat viral infections.
  • compositions and methods for inactivating viruses and retroviruses present in blood, other body fluids and, more generally, biological fluids include the antiviral compounds hypericin,
  • compositions for treating retroviral infections with hypericin, pseudohypericin, salts and mixtures with nucleoside analogues, such as AZT disclose compositions for treating retroviral infections with hypericin, pseudohypericin, salts and mixtures with nucleoside analogues, such as AZT.
  • hypericin obtained from plants of the genus Hypericum or prepared synthetically, varied in their biological activity. Also the hypericin preparations differed in some of their physical properties, including solubility in organic solvents and formation of dispersions in water. This variation apparently resulted from the way hypericin was extracted and purified. Therefore, the need arose to find compounds possessing a hypericin moiety which would give consistent antiviral and
  • the present invention relates to novel compounds possessing a hypericin moiety consisting of ion pairs having anti- viral activity.
  • ion pairs consisting of negatively charged hypericin ions bound to cationic species, either organic or inorganic, are useful in the treatment of various diseases caused by viruses and particularly retroviruses in humans and animals and are also useful in neutralizing the infectivity of viruses in vitro .
  • the ion pairs can be prepared by first treating the hypericin with acid to yield free hypericin. Free hypericin is then dispersed in an organic or inorganic base to form an ion pair.
  • the hypericin ion pair may be used for the prophylactic or therapeutic treatment of an individual exposed to or suspected of being infected with a virus.
  • the hypericin ion pair may also be used for inhibiting the growth of a virus in vi tro or disinfecting fluids and materials.
  • Fig. 1 is a graph plotting the percent inhibition of Friend virus induced splenomegaly in mice as a function of the administered amount of hypericin-sodium ion pair and hypericin-lysine ion pair.
  • Fig. 2 is a graph plotting the percent inhibition of Friend virus induced splenomegaly in mice as a function of the administered amount of hypericin-sodium ion pair and hypericin-Tris ion pair.
  • Fig. 3 is a graph plotting the percent inhibition of Friend virus induced splenomegaly in mice as a function of the administered amount of hypericin-sodium ion pair and hypericin-ammonium ion pair.
  • Fig. 4 is a graph plotting the antiretroviral activity of various hypericin ion pairs by monitoring the direct inactivation of murine radiation leukemia virus as measured by the inhibition of virus-particle derived reverse transcriptase activity.
  • hypericin which has heretofore been obtained by isolation from plants contains a certain amount of sodium ions.
  • the usual way to isolate hypericin from plant material is by extraction with polar solvents such as ethanol, methanol, acetone and the like, followed by chromatography on silica gel.
  • this form of hypericin has sodium ions originating from silica gel which invariably contains sodium ions.
  • the relative quantities of sodium ions in the pure hypericin obtained by chromatography on silica gel may vary, depending on the type of the silica gel used, the solvent applied in the elution procedure, and the rate of the elution.
  • the hypericin prepared by chemical synthesis contains sodium ions, since generally its purification also employs chromatography on silica gel.
  • hypericin in the pure form described in the above-cited Merck Index and in the literature is crystallization or precipitation from pyridine solutions in the absence or in the presence of hydrogen chloride. These crystals are very slightly soluble in organic solvents, and practically insoluble in water. These may be converted to a soluble form of hypericin also by chromatography on silica gel.
  • the present inventors have discovered by means of X-ray diffraction that such crystals of hypericin precipitated from pyridine solutions contain two molecules of pyridine and one of water. According to the diffrac tion pattern, one of the hydrogen atoms of the two
  • the compound is first acidified to form free hypericin (I) in which none of the hydroxy groups are ionized and any pyridine or sodium paired to the molecule during work-up is removed.
  • Free hypericin is acidic and has different IR, UV-visible and fluorescent spectra characteristics than are obtained from the starting form of hypericin.
  • Hypericin ion pairs differ from hypericin salts, the green colored material described above, by their solubility in organic polar solvents, their color and their very low electric conductance. Thus, the ion pairs behave in all respects as organic compounds and can be defined as such and not as salts.
  • a compound having a structure of improved predictability and reproducibility is formed with improved predictability and reproducibility with respect to the properties of solubility and biological activity.
  • the hypericin ion pairs of the present invention have the general formula:
  • X is a monovalent cation and n is 1, or X is a divalent cation and n is 2, X not being a sodium or pyridinium cation.
  • the present invention comprehends the use of any organic or inorganic base, with the exception of NaOH and pyridine, as the cation in the ion pair as long as the pH is not such as to create the divalent salt of hypericin.
  • Preferred inorganic cationic species include the alkali and alkaline earth metal cations, such as potassium, lithium, calcium and magnesium, as well as ammonium.
  • Preferred organic cations are protonated nitrogen base moieties of the general formula NR 1 R 2 R 3 R 4 where R 1 -R 4 are the same or different and may be hydrogen;
  • the cation may be any of the basic amino acids.
  • Two of the R groups may, together with the nitrogen atom to which they are attached, form a
  • pyrrolidine piperidine, morpholine, piperazine or an N-alkyl substituted piperazine ring, or three of the R groups form together with the nitrogen atom to which they are attached an alkyl-substituted pyridine ring.
  • the various ion pairs of the present invention have varying solubility in water depending on the cation which is chosen.
  • the preferred organic cationic species are those of the general formula, NHR 1 R 2 R 3 in which R 1 -R 3 are all ethyl or 2-hydroxyethyl; R 1 and R 2 are hydrogen and R 3 is octyl, 2-hydroxyethyl, 2-aminoethyl, 5-amino-5-carboxypentyl (lysine) or 1, 3-dihydroxy-2-hydroxymethyl-2propyl; R 1 is hydrogen and R 2 and R 3 are 2-hydroxyethyl or 2-hydroxypropyl; or R 1 is hydrogen, R 2 is methyl and R 3 is 1-desoxy-1-sorbityl.
  • Compounds wherein X is an inorganic monovalent or divalent cation may be produced by addition of the free hypericin to a water solution or suspension of the corresponding inorganic base such as alkali carbonate, bicarbonate, hydroxide, phosphate, etc.
  • a water solution or suspension of the corresponding inorganic base such as alkali carbonate, bicarbonate, hydroxide, phosphate, etc.
  • compounds wherein X is ammonium, lithium or potassium cations may be prepared by addition of free hypericin to a water solution of ammonium hydroxide, lithium hydroxide or potassium hydroxide, respectively, preferably using one equivalent, followed by lyophilization of the resulting solution to dryness.
  • the ion pairs may be prepared by addition of free hypericin to a water solution of calcium hydroxide or magnesium hydroxide, respectively, preferably using half an equivalent, followed by
  • the hypericin ion pairs wherein X is an organic cation derived from a nitrogen base bound to hydrogen of the structure NH 1 R 2 R 3 may be prepared by treating free hypericin dissolved in an organic polar solvent with the respective nitrogen base, preferably using one equivalent. Alternatively, a suspension of the free hypericin in water may be treated with the nitrogen base.
  • the hypericin ion pair may be prepared by addition of free hypericin to an ethanol solution of triethanolamine, followed by evaporation to dryness.
  • N + (R 1 , R 2 , R 3 ,R 4 ) may be prepared by treating a hypericin ion-pair, preferably containing inorganic monovalent cationic species which may include sodium, potassium, ammonium or lithium cations dissolved in an organic polar solvent or water, with the respective quaternary ammonium salt.
  • a hypericin ion-pair preferably containing inorganic monovalent cationic species which may include sodium, potassium, ammonium or lithium cations dissolved in an organic polar solvent or water, with the respective quaternary ammonium salt.
  • the hypericin ion pair wherein X is
  • the hypericin ion pairs of the present invention are characterized by their solubility in organic polar solvents. Some of these derivatives can be dispersed in water, forming high molecular weight, loosely associated polymeric structures containing occluded water which can be decomposed back to the monomeric hypericin by addition of organic solvents.
  • hypericin ion-pairs in water dispersions, hypericin ion-pairs are in an aggregate state, but inside cellular or viral membranes they exist in a monomeric form. Thus, the bioabsorption of the hypericin ion-pairs necessitates dissociation of the aggregates into monomeric molecules. This slow down in the bioabsorption effect will provide slow-release of active substances. It is to be anticipated that in such cases a constant level of the active substances in the body will be kept for comparatively long periods.
  • Liposomes are a model for all membranes. High solubility in liposomes is indicative that the compound enters the cell rapidly and thus more frequent doses have to be administered, while low solubility indicates that the compound enters the cell slowly. Thus, amounts of the compound remain in the organism and act as slow release agents, thus necessitating less frequent administration of doses.
  • Solubility of hypericin ion-pairs in liposomes is given in Table 1.
  • a solution of liposomes was prepared from lecithin (10%). This solution was mixed with an equal volume of water containing various hypericin ion-pairs. The maximum binding capacity of the liposomes for the following hypericin ion-pairs was established.
  • hypericin-lysine and hypericin-ethylene diamine ion-pair act as slow release vehicles for the target organs is shown by their comparative low solubility in liposomes (as compared with the solubility of sodium and lithium ion-pairs). However, all ion-pairs would be expected to be less soluble than salts which are fully dissolved in monomeric form.
  • antiviral activity against Friend virus has been measured using ion pairs of hypericin with sodium, lysine, ethylenediamine, diethanolamine, N-methylglucamine, triethanolamine, Tris and ammonium.
  • the hypericin ion pairs of the present invention can be advantageously used as prophylactic antiviral agents, for treatment of virally-infected mammals or for inactivating viruses and retroviruses present in biological fluid as in U.S. Patent No. 5,149,718.
  • the effectiveness of hypericin and pseudohypericin for antiviral effects has been shown in U.S. Patent 4,898,891.
  • the hypericin ion pairs of the invention can be combined for antiviral therapy with nucleoside analogs such as
  • hypericin ion pairs of the invention may be used with other antiviral agents such as adamantine, rifampicin, or with vaccines or antibody preparations.
  • "In conjunction" includes successive administration, co-administration, substantially contemporaneous administration of different preparations or alternating administration of nucleoside analog therapy and hypericin ion pairs.
  • the hypericin ion pairs may be used in conjunction with other biologically effecting substances.
  • Other known antibiotics may be used in conjunction with
  • hypericin ion pairs even if that antibiotic does not have any effect on viral growth per se.
  • hypericin ion pair containing compositions may include one or more immunomodulating chemicals.
  • Biological materials and transplants may also contain hypericin ion pairs as part of the therapy.
  • the hypericin ion pairs of the present invention in their water-dispersed or water-solubilized forms have a wide spectrum of effectiveness in inhibiting viruses and retroviruses.
  • viruses which are inhibited by the compounds of the present invention are cytomegalovirus, Herpes Simplex Virus (HSV), influenza virus, Vesicular Stomatitis Virus (VSV), Hepatitis B virus, papilloma virus and retroviruses, such as HIV, HTLV I, HTLV II and feline leukemia virus.
  • Effective inhibition of a given virus may be achieved by using one of the compounds of the present invention or a combination of two or more of such compounds.
  • a single hypericin ion pair may constitute the sole active ingredient of the compositions of the present invention or may be employed in conjunction with other antiviral agents acting by any means .
  • the determination of the most effective compound or mixture of compounds and the concentration of effective treatment of the particular virus or retrovirus responsible for the infection can be ascertained by routine experimentation using suitable experimental models well-known in the art.
  • the hypericin ion pairs of the present invention may be administered orally, topically or parenterally, and preferably intravenously at dosages broadly ranging between about 0.0001 micrograms and about 100,000 micrograms per kilogram (kg) of body weight of the treated mammal per treatment. More preferably, the dosage range may between about 0.1 and about 50,000 micrograms per kg body weight of the treated mammal per treatment.
  • the hypericin ion pairs of the present invention may also be used on various surfaces to inactivate or inhibit viruses in vitro.
  • the surfaces to be treated include surgical equipment, prosthetic devices, interior and/or exterior surfaces of gloves, condoms, body fluid handling devices such as needles, syringes, cathe ters, etc.
  • the compositions of the invention may be used as antiseptics broadly as well to disinfect organs, fluids, skin, table tops, containers, equipment, etc. A thin film of the composition may remain on the surface.
  • the present invention also provides pharmaceutical compositions and formulations for treating viral infections.
  • the hypericin ion pairs of the present invention can be incorporated in conventional solid or liquid pharmaceutical formulations in any concentration desired.
  • tablets, capsules, caplets, injectable or orally administrable solutions may be used for treating mammals that are afflicted with viral infections.
  • the pharmaceutical formulations of the invention comprise an effective amount of the hypericin ion pair of the present invention as the active ingredients. Other active or inert ingredients may be added.
  • a parenteral therapeutic composition may comprise a sterile isotonic saline solution containing between 0.001 micrograms of the hypericin ion pair of the present invention. It will be appreciated that the unit content of active ingredients contained in an individual dose of each dosage form need not in itself constitute an effective amount provided that the effective amount can be reached by administration of a plurality of doses.
  • Each formulation according to the present invention may additionally comprise inert constituents including pharmaceutically-acceptable carriers, diluents, fillers, salts, and other materials well-known in the art, the selection of which depends upon the dosage form utilized, the particular purpose to be achieved according to the determination of the ordinarily skilled artisan in the field and the properties of such additives.
  • carriers and diluents include carbohydrates and lipids including, without limitation, phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, triglycerides, tocopherol, retinoic acid, cyclodextrins and their derivatives.
  • Hypericin ion pairs of the present invention may additionally be incorporated into transdermal delivery systems or liposomes, the latter for use as specific drug carriers.
  • liposomes may also comprise other active agents, e.g., specific anti-HIV antibodies directed against viral proteins expressed by virally infected cells such as HIV gp120, p120, gp41, p 41 and p24 to act as specific targeting agents.
  • Topical use of the hypericin ion pairs in a suitable carrier may also be used. This may be of particular importance in viral rashes, herpes simplex vesicles (type I or type II), shingles, etc.
  • hypericin as the compound which is formed into an ion pair
  • this ion pair technology is also applicable to other polycyclic aromatic diones which have antiviral and antiretroviral activity and which have two reactive hydroxyl groups in their free acid form as does hypericin.
  • pseudohypericin has the same structure as hypericin except that one of the two methyl groups shown on the right-hand side of formula (I) is replaced by CH 2 OH.
  • substituents of hypericin may also be made on moieties other than the hydroxy groups on which the ion pair is formed, so long as such modifications do not affect the solubility properties of the ion pairs and do not substantially affect the antiviral and antiretroviral properties of the compounds themselves.
  • These compounds will be referred to herein as hypericin analogs and derivatives.
  • the methyl groups may be replaced by other alkyl, alkylamino, alkoxy, and alkylaminoalkyl groups. These two groups may be combined to form a six or seven membered ring.
  • the remaining four hydroxy groups on the hypericin molecule may be eliminated (H) or substituted with an ether group (OR) or an amino group (NRR) in which R may be hydrogen or alkyl.
  • R may be hydrogen or alkyl.
  • R may be hydrogen or alkyl.
  • R may be hydrogen or alkyl.
  • R may be hydrogen or alkyl.
  • the hypericin used in the following examples was prepared according to the methods in U.S. Patent No.
  • Free hypericin was prepared by mixing a solution of hypericin (1g) in acetone (150 mL) and treating it with hydrochloric acid solution 7% (5 mL). The resulting brown precipitate was filtered and washed with water until it became neutral. The precipitate was collected and dried in high vacuum at 70°C.
  • IR spectrum (KBr) showed adsorption bands at 3400, 1625, 1598, 1473, 1426, 1399, 1369, 1330, 1300, 1224, 1188, 1153, 1116, 1090, 844, 806, 794, 673 cm -1 .
  • Hypericin sodium ion pair was prepared by slowly adding powdered free hypericin (0.25g), obtained in Example 1, to a stirred solution of acetone (50 ml) and 5% aqueous sodium bicarbonate (168 ml, 2mM). After about 0.5 hours, the resulting solution was filtered and the fil trate evaporated to dryness. The ion pair was further dried in high vacuum at 70°C.
  • IR spectrum (KBr) showed adsorption bands at 3400, 1615, 1590, 1559, 1500, 1465, 1422, 1353, 1386, 1337, 1260, 1187, 1114, 843, 805, 685, 604, 567, 538, 527 cm -1
  • Hypericin potassium ion pair was prepared by slowly adding powered free hypericin (0.25g), obtained in Example 1, to a stirred solution of acetone (50 ml) and 2% aqueous potassium hydroxide (5.6 ml, 2mM). After about
  • Hypericin lithium ion pair was prepared by slowly adding powdered free hypericin (0.25g), obtained in Example 1, to a stirred solution of acetone (50 ml) and 0.3 ml of 3% aqueous lithium hydroxide monohydrate (2mM). After about 0.5 hours, the resulting solution was filtered and the filtrate evaporated to dryness. The ion pair was further dried under high vacuum at 70°C. IR spectrum (KBr) showed adsorption bands at 3400, 1590, 1558, 1501, 1465, 1422, 1393, 1367, 1336, 1288, 1260, 1188, 1113, 843, 666, 625, 603, 567, 528 cm -1
  • Hypericin ammonium ion pair was prepared by slowly adding powdered free hypericin (0.25g), obtained in Example 1, to a stirred solution of acetone (50ml) and 0.32 ml of 5% ammonium bicarbonate (2 mM). After about 0.5 hours, the resulting solution was filtered and the filtrate evaporated to dryness. The ion pair was further dried under high vacuum at 70°C.
  • IR spectrum (KBr) showed adsorption bands at 3400, 3200, 1620, 1585, 1556, 1499, 1461, 1418, 1365, 1334, 1260, 1183, 1114, 893, 850, 831, 804, 671, 624 cm -1
  • UV visible absorption spectrum ⁇ max 590, 547, 509, 474 nm
  • Hypericin calcium ion pair was prepared by slowly adding free hypericin (0.25g), obtained in Example 1, to a stirred solution of acetone (50 ml) and 1 ml at 2% aqueous calcium hydroxide. After about 0.5 hours, the resulting solution was filtered and the filtrate evaporated to dryness. The ion pair was further dried under high vacuum at 70°C.
  • IR spectrum (KBr) showed adsorption bands at 3500, 1620, 1585, 1563, 1490, 1463, 1418, 1386, 1334, 1260, 1180, 1116, 845, 806, 677, 625, 604, 571 cm -1
  • Hypericin magnesium ion pair was prepared by slowly adding powdered free hypericin (0.25g), obtained in Example 1, to a stirred solution of acetone (50 ml) and 7 mL of magnesium hydroxide pentahydrate in 2% aqueous basic magnesium carbonate. After about 0.5 hours, the resulting solution was filtered and the filtrate evaporated to dryness. The ion pair was further dried under high vacuum at 70°C.
  • IR spectrum (KBr) showed adsorption bands at 3500, 1620, 1585, 1563, 1490, 1463, 1418, 1386, 1334, 1260, 1180, 1116, 845, 806, 677, 625, 604, 571 cm -1
  • Hypericin triethylammonium ion pair was prepared by mixing a solution of triethylamine (0.05g) in acetone (50mL), treating it with free hypericin powder (0.25g), obtained in Example 1, and then stirring at room temperature for 30 minutes. The resulting dark red solution was evaporated in vacuum to dryness.
  • Hypericin octylammonium ion pair was prepared by mixing a solution of octylamine (0.065g) in acetone (50 mL), treating it with free hypericin powder (0.25g), obtained in Example 1, and then stirring at room temperature for 30 minutes. The resulting dark red solution was evaporated in vacuum to dryness.
  • UV visible absorption spectrum ⁇ max 590, 547, 509, 474 nm
  • Hypericin 2-hydroxyethylammonium ion pair was prepared by mixing ethanolamine (0.03g) in acetone (50 mL), treating it with free hypericin powder (0.25g), obtained in Example 1, and then stirring at room temperature for 30 minutes. The resulting dark red solution was evaporated in vacuum to dryness.
  • UV visible absorption spectrum ⁇ max 590, 547, 509, 474 nm
  • Hypericin 2-amino-ethylammonium ion pair was prepared by mixing ethylenediamine (0.037g) in acetone (50 mL), treating it with free hypericin powder (0.25g), obtained in Example 1, and then stirring at room temperature for 30 minutes. The resulting dark red solution was evaporated in vacuum to dryness.
  • UV visible absorption spectrum ⁇ max 590, 547, 509, 474 nm
  • Hypericin bis-(2-hydroxyethyl) ammonium ion pair was prepared by mixing diethanolamine (0.05g) in acetone (50 mL), treating with free hypericin powder (0.25g), obtained in Example 1, and then stirring at room temperature for 30 minutes. The resulting dark red solution was evaporated in vacuum to dryness.
  • Hypericin bis-(2-hydroxypropane) ammonium ion pair was prepared by mixing diisopropanolamine (0.05g) in acetone (50 mL), treating with free hypericin powder
  • Example 2 (0.25g), obtained in Example 1, and then stirring at room temperature for 30 minutes. The resulting dark red solution was evaporated in vacuum to dryness.
  • UV visible absorption spectrum ⁇ max 589, 547, 509, 473 nm
  • Hypericin tris-(2-hydroxyethyl)ammonium ion pair was prepared by mixing triethanolamine (0.075g) in acetone (50 mL), treating with free hypericin powder (0.25g), obtained in Example 1, and stirring at room temperature for 30 minutes. The resulting dark red solution was evaporated in vacuum to dryness.
  • Hypericin lysinium ion pair was prepared by mixing lysine (0.08g) in water (20 mL), treating with free hypericin powder (0.25g), obtained in Example 1, and stirring at room temperature overnight. The resulting solution was lyophilized until dry.
  • IR spectrum (KBr) showed adsorption bands at 3500, 3100, 3000, 1618, 1585, 1502, 1482, 1419, 1386, 1335, 1259, 1182, 1114, 839, 804, 675, 666 cm -1
  • Hypericin N-methylglucammonium ion pair was prepared by mixing N-methylglucamine (0.2g) in water (20 mL), treating with free hypericin powder (0.25g), obtained in Example 1, and stirring at room temperature overnight.
  • the resulting solution was lyophilized until dry.
  • Hypericin Tris ion pair was prepared by mixing
  • EXAMPLE 18 Hypericin-tetramethylammonium ion pair.
  • EXAMPLE 21 The Antiretroviral Activity of Hypericin- Lysine Ion Pair against Murine Friend Virus in BALB/c mice.
  • mice infected with Friend Virus develop a virus-induced erythroleukemia which results in death of 100% of the animals within 25-45 days.
  • An early manifestation of the infection is a large increase in spleen size
  • mice in groups of 3 were infected with Friend virus and
  • hypericin-lysine ion pair was then administered intravenously, within one hour of the infection, in a single dose of 1, 10, 50 and 150 micrograms/mouse.
  • 0.5 ml solution of the compound in PBS was administered.
  • Standard hypericin obtained by a process which includes chromatography on silica gel and thus is a sodium ion pair
  • solubilized in 0.5% aqueous benzyl alcohol and administered at the same concentrations was used as a control, along with normal control mice infected with PBS. All mice were sacrificed after 10 days and analyzed for spleen weights.
  • the results, shown in Fig. 1, show that both hypericin-lysine and hypericin-sodium inhibited splenomegaly of Friend Virus infected mice by approximately 70-80% at dose ranges of 10-50 micrograms/mouse.
  • EXAMPLE 23 The Antiretroviral Activity of Hypericin- Anmonium Ion Pair against Murine Friend Virus in BALB/c Mice
  • EXAMPLE 24 Determination of the Antiretroviral Activity of Various Hypericin Ion Pairs by Monitoring the Direct Inactivation of Murine Radiation Leukemia Virus as Measured by the Inhibition of Virus-Particle Derived Reverse Transcriptase Activity
  • eda hypericin-ethylenediamine
  • tea hypericin-triethanalamine
  • dpa hypericin-diisopropanolamine
  • nmg hypericin-n-methylglucamine
  • Fig. 4 are expressed as count per minute of 3 H-thymidine incorporation. They show significant inhibition of virus- particle derived reverse transcriptase with eda being the least efficacious. See Fig. 4.
  • test compounds The antiviral activity of the test compounds was assayed at final concentrations of 32, 10, 3.2, 1.0, 0.32 and 0.1 ⁇ g/ml.
  • Stock solutions were prepared by dissolving a preweighed amount of test compound in
  • DMSO dimethylsulfoxide
  • Antiviral activity was evaluated in mouse mammary tumor fibroblasts (C127L) grown in 24-well tissue culture plates. Four wells were used for each concentration of each test article. After aspirating the medium from the tissue culture plates, three of the wells received 0.5 ml of virus inoculum; the fourth well received 0.5 ml of 2% MM. The plates were incubated for 1 hr at 37°C in 5% CO 2 with continuous agitation. After incubation, the virus inoculum was removed and an agar overlay added. A separate overlay was made for each concentration of test compound. Each overlay consisted of 3 ml of test compound (prepared as a 2x solution) plus 3 ml of agar overlay. After mixing, 1 ml of the combination overlay was added to each of the four wells, the fourth well being the test compound toxicity control. Cell control and virus control wells were included on each plate.
  • Acyclovir, the positive antiviral control, and a virus titration were run simultaneously with the test.
  • the plates were incubated at 37°C in 5% CO 2 for six days, after which time the overlay was removed and the plates were fixed and stained with 1% crystal violet.
  • the plaques were read using an inverted microscope and the number of plaques in each well was recorded.
  • the average plaque forming units (PFU) per ml and percent reduction results from this in vitro assay are shown in Table 2, with the plaque counts for individual wells shown in Table 3.
  • a test compound concentration resulting in a reduction of 50% or more in the number of plaques as compared to the virus control ( ⁇ 44 PFU/ml) is an indication of good antiviral activity.
  • Hypericin was toxic at both the 32 and 10 ⁇ g/ml concentrations, but showed a 51% plaque reduction at 3.2 ⁇ g/ml, with no reduction at any of the other concentrations tested. Both hypericin ion pairs were toxic at 32 ⁇ g/ml and moderately toxic at the 10 ⁇ g/ml concentration.
  • the N-methylglucamine derivative even though moderately toxic at 10 ⁇ g/ml, showed an 88% reduction in plaques, with no reduction in plaques at any of the other concentrations.
  • the lysine derivative showed a 100% reduction in plaques at the 10 ⁇ g/ml concentration. Only 25% of the cell sheet was still attached when the assay was read, but these cells were normal and there were no plaques present. None of the other concentrations tested showed any signs of reduction.
  • Acyclovir the positive antiviral control, showed complete protection at all of the concentrations tested.
  • Results are shown for the individual assay wells at each drug concentration. The mean and standard deviation for thirty untreated, virus
  • control wells was 87 ⁇ 15.3 PFU/ml.
  • hypericin was toxic at the 32 and 10 ⁇ g/ml concentrations, but showed a 51% reduction of plaques at the 3.2 ⁇ g/ml concentration. Both hypericin derivatives were toxic at the 32 ⁇ g/ml concentration, and, even though both were moderately toxic at the 10 ⁇ g/ml concentration, the N-methylglucamine derivative showed a plaque reduction of 88% and the lysine derivative showed a plaque reduction of 100%.
  • Example 26 In Vivo Assay of Hypericin and Two Ion Pairs thereof against MCMV
  • mice For the in vivo evaluation of the three test articles against MCMV, dosing concentrations of 50, 25, and 10 mg/kg body weight (mpk) were used. Virus antibody-free four week old female CDl mice were obtained from Charles River Breeding Laboratories (Portage, Michigan). The animals were randomly housed five per cage after being examined grossly for general activity and alertness. The mice were quarantined for twelve days prior to dosing and were provided with food and water ad libitum throughout the quarantine period and the study. Three days before dosing began, the animals were weighed and assigned to groups with dosages being determined on a group weight basis, with each group consisting of ten mice. The Groups were assigned as follows:
  • All dosing solutions were prepared at concentrations such that the desired dosage of compound could be administered in a volume of 0.2 ml.
  • the three test compounds were dissolved in sterile water at concentrations of 5.75 mg/ml for the 50 mpk dosing solutions.
  • hypericin 5.75 mg/ml for the N-methylglucamine derivative, and 5.5 mg/ml for the lysine derivative.
  • the 25 and 10 mpk dosing solutions were prepared by dilution with sterile water from the 50 mpk concentration.
  • mice were administered the test articles in a volume of 0.2 ml by intraperitoneal injection once each day for eight consecutive days.
  • the vehicle control group received daily injections of 0.2 ml of sterile water.
  • On the second day approximately five hours after receiving the second dosing of test compound, all mice were administered a 0.2 ml challenge dose of MCMV by intraperitoneal injection. Mice were observed for mortality for twentyone days after virus challenge. Percent mortality and mean survival times (MST) were calculated for each group.
  • Treatment with hypericin at the 25 and 50 mpk concentrations reduced mortality to 10% and 0%, respectively, while the mean survival times increased to 19.7 and 21 days.
  • the survival of animals treated with hypericin at 10 mpk was not significantly different from the vehicle control group.
  • the N-methylglucamine ion pair derivative showed reduced mortality and an increased MST at all three concentrations.
  • the 10, 25, and 50 mpk concentrations resuited in mean survival times of 18, 21, and 21 days, with mortalities of 20%, 0%, and 0%, respectively.
  • the lysine ion pair derivative reduced mortality at the 25 and 50 mpk concentrations to 20% and 0%, respectively, with increased MST to 18 and 21 days. Survival of the 10 mpk group was not significantly different from that of the vehicle control group.
  • For the positive control compounds For the positive control compounds,
  • Acyclovir treatment at 25 mpk resulted in a MST of 19.4 days with a mortality of 10%, while Ara- A at 25 mpk produced a MST of 21 days with a mortality of 0%.

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Abstract

On a préparé des paires d'ions d'hypericine ainsi que de ses analogues et dérivés, par acidification d'hypericine jusqu'à obtention de sa forme exempte d'acide, et par réaction avec une quantité prédéterminée d'une base organique ou inorganique à un pH inférieur à environ 11,5. Les composés sont utiles comme agents thérapeutiques et antiseptiques antiviraux.
PCT/US1993/001393 1992-02-14 1993-02-16 Paires d'ions de composes d'hypericine ayant une activite antivirale WO1993015607A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP5514335A JPH07504170A (ja) 1992-02-14 1993-02-16 抗ウイルス性活性を有するヒペリシン化合物のイオン対
AU37205/93A AU679676B2 (en) 1992-02-14 1993-02-16 Ion pairs of hypericin compounds having antiviral activity
EP93906002A EP0644869A4 (en) 1992-02-14 1993-02-16 Ion pairs of hypericin compounds having antiviral activity.

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US83589692A 1992-02-14 1992-02-14
US835,896 1992-02-14

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JP (1) JPH07504170A (fr)
AU (1) AU679676B2 (fr)
CA (1) CA2130090A1 (fr)
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WO (1) WO1993015607A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6229048B1 (en) 1997-07-31 2001-05-08 Yeda Research And Development Co. Ltd. At The Weizmann Institute Of Science Helianthrone derivatives as anti-cancer agents
WO2008087441A2 (fr) * 2007-01-19 2008-07-24 Ultra Biotech Limited Composition de prévention et de traitement de la grippe
WO2011104667A1 (fr) * 2010-02-25 2011-09-01 Anthem Biosciences Private Limited Sels d'acides aminés basiques de polyphénols
WO2017151111A1 (fr) 2016-03-01 2017-09-08 Soligenix, Inc. Systèmes et procédés pour produire de l'hypéricine synthétique

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4898891A (en) * 1987-08-07 1990-02-06 Yeda Research And Development Company Ltd. Antiviral compositions
AU3056492A (en) * 1991-11-01 1993-06-07 New York University Hypericin compositions for treating t-cell mediated diseases

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
Organic Chemistry, 1983, Morrison and Boyd, pp. 821-22 and 960, 966, and 970-72. *
Physical Pharmacy, 1983, MARTIN et al., pp. 588-78. *
Proceedings of the National Academy of Sciences, August 1989, LAVIE et al., "Studies of the Mechanisms of Action of the Antiretroviral Agents Hypericin and Pseudohypericin", pp. 5963-67, entire article. *
Proceedings the National Academy of Sciences, July 1988, MERUELO et al., "Therapeutic Agents with Dramatic Antiretroviral Activity and Little Toxicity at Effective Doses: Aromatic Polycyclic Diones Hypericin and Pseudohypericin", pp. 5230-5234, see entire article. *
Remington's Pharmaceutical Sciences, 1975, 1001-02. *
See also references of EP0644869A4 *
The Merk Index, 1983, Wind Holz (ed.), pg. 710. *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6229048B1 (en) 1997-07-31 2001-05-08 Yeda Research And Development Co. Ltd. At The Weizmann Institute Of Science Helianthrone derivatives as anti-cancer agents
WO2008087441A2 (fr) * 2007-01-19 2008-07-24 Ultra Biotech Limited Composition de prévention et de traitement de la grippe
WO2008087441A3 (fr) * 2007-01-19 2008-09-18 Ultra Biotech Ltd Composition de prévention et de traitement de la grippe
WO2011104667A1 (fr) * 2010-02-25 2011-09-01 Anthem Biosciences Private Limited Sels d'acides aminés basiques de polyphénols
WO2017151111A1 (fr) 2016-03-01 2017-09-08 Soligenix, Inc. Systèmes et procédés pour produire de l'hypéricine synthétique
CN109071397A (zh) * 2016-03-01 2018-12-21 索利吉尼克斯公司 用于生产合成金丝桃素的系统和方法
EP3423428A4 (fr) * 2016-03-01 2019-12-11 Soligenix, Inc. Systèmes et procédés pour produire de l'hypéricine synthétique
AU2016396033B2 (en) * 2016-03-01 2021-07-08 Soligenix, Inc. Systems and methods for producing synthetic hypericin
AU2021203704B2 (en) * 2016-03-01 2023-02-16 Soligenix, Inc. Systems and methods for producing synthetic hypericin

Also Published As

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IL104693A0 (en) 1993-06-10
EP0644869A1 (fr) 1995-03-29
AU3720593A (en) 1993-09-03
AU679676B2 (en) 1997-07-10
EP0644869A4 (en) 1995-04-12
CA2130090A1 (fr) 1993-08-19
JPH07504170A (ja) 1995-05-11

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